One of the least understood and most complex disease processes is the transformation that occurs as a cell becomes malignant. This process involves both genetic mutations and proteomic transformations, the result of which, allows the cell to escape normal controls; preventing inappropriate cell division. Cancer cells share some common attributes. Most cancer cells proliferate outside of the normal cell cycle controls, exhibit morphological changes and exhibit various biochemical disruptions to cellular processes.
Cancer is usually diagnosed when a tumor becomes visible well after the first on-set of cellular changes. Many cancers are diagnosed after a biopsy sample is examined by histology for morphologic abnormalities, evidence of cell proliferation and genetic irregularities. Effective treatment for malignancy often depends on the ability to detect reliably, the presence of malignant cells at early stages of a disease so that an effective treatment can begin at a stage when the disease is more susceptible to such treatment. Thus, there is a need to be able to reliably detect a potentially malignant cell that has not progressed to the histological stage recognized as malignant, but which can progress to a malignant state. There is also a need for a rapid, minimally invasive technique that can reliably detect or treat malignant cells or potentially malignant cells.
Proliferating cell nuclear antigen (PCNA) is a 29 kDa nuclear protein and its expression in cells during the S and G2 phases of the cell cycle, makes the protein a good cell proliferation marker. It has also been shown to partner in many of the molecular pathways responsible for the life and death of the cell. Its periodic appearance in S phase nuclei suggested an involvement in DNA replication. PCNA was later identified as a DNA polymerase accessory factor in mammalian cells and an essential factor for SV40 DNA replication in vitro. In addition to functioning as a DNA sliding clamp protein and a DNA polymerase accessory factor in mammalian cells, PCNA interacts with a number of other proteins involved in transcription, cell cycle checkpoints, chromatin remodeling, recombination, apoptosis, and other forms of DNA repair. Besides being diverse in action, PCNA's many binding partners are linked by their contributions to the precise inheritance of cellular functions by each new generation of cells. PCNA may act as a master molecule that coordinates chromosome processing.
PCNA is also known to interact with other factors like FEN-1, DNA ligase, and DNA methyl transferase. Additionally, PCNA was also shown to be an essential player in multiple DNA repair pathways. Interactions with proteins like the mismatch recognition protein, Msh2, and the nucleotide excision repair endonuclease, XPG, have implicated PCNA in processes distinct from DNA synthesis. Interactions with multiple partners generally rely on mechanisms that enable PCNA to selectively interact in an ordered and energetically favorable way.
Clues to a mechanism of PCNA's functions were initially uncovered through investigation of the DNA synthesome, a multiprotein DNA replication complex present in mammalian cells. Studies examining the synthetic activity of the DNA synthesome identified an increased error rate in malignant cells when compared to non-malignant cells. These results suggest that a structural alteration to one or more components of the DNA synthesome in malignant cells has occurred. 2D-PAGE immunoblot analysis of PCNA, an essential component of the DNA synthesome, revealed two distinct isoforms with vastly different isoelectric points (pIs). One PCNA isoform displayed a significantly basic pI and was present in both malignant and non-malignant cells. The other isoform had an acidic pI and was found exclusively in malignant cells. Because of its presence only in malignant cells, this isoform was termed the cancer-specific isoform or csPCNA, and the post-translational alteration that is responsible for PCNA's altered 2D-PAGE migration pattern remains undetermined.
Some labeling studies with PCNA suggested that the migration of PCNA was most likely not due to alterations such as phosphorylation, acetylation, glycosylation, or sialyzation. Conflicting studies have surfaced attempting to identify post-translational modifications to PCNA. For example, the phosphorylation of PCNA was reported to affect its binding to sites of DNA synthesis. Another study claimed that PCNA was, after all, not phosphorylated but acetylated. In addition to these studies, analysis of yeast PCNA has shown it to be the target of ubiquitination in response to DNA damage and sumoylation in the absence of damage. Due to the diverse and conflicting structural evidence for PCNA, it is difficult to identify which modifications, if any, are responsible for the appearance and functions of csPCNA isoform.
Therefore, identification of the correct post-translational modifications of csPCNA is desirable to develop diagnostic methods and also to develop therapeutics based on the interactions of csPCNA with its partners. Malignant cancer cells express an isoform of PCNA termed cancer specific PCNA (csPCNA) and non-malignant cells express an isoform termed non-malignant PCNA (nmPCNA). Effective compositions and methods to distinguish the two isoforms are needed for diagnosis and treatment of cancers.